Gene Transformation Of Streptomyces Ahygroscopicus, Cloning And Function Of Cytosinpeptidemycin Biosynthetic Genes

Cytosinpeptidemycin is a newly reported antiphytoviral agricultural antibiotic produced by Streptomyces ahygroscopicus var.Liaoningensis,which owns high control effect to some plant viral and fungal diseases.To study the biosynthesis of cytosinpeptidemycin in the view of molecular biology,the gene transformation system of Streptomyces ahygroscopicus was developed,some biosynthetic gene was cloned and analyzed in the study.The results are as fellows:1.Wild strain of Streptomcyes ahygroscopicus was mutagenized by UV irradiation.A matant,UV2136,blocked in the biosynthesis of cytosinpeptidemycin was used to clone cytosinpeptidemycin biosynthetic genes.Meanwhile,a high-producing strain was induced, which can be used in the cytosinpeptidemycin production.2.Targeted with UV2136,the mutant of Streptomyces ahygroscopicus var.Liaoningensis blocked in cytosinpeptidemycin biosynthesis,the optimal conditions for mycelium growth, protoplasts formation and regeneration were developed.Exquisite and abundant mycelium was typically grown in 40ml of adopted YEME medium containing 15%of glucose and 0.5% of glycine.The optimal lysozyme system,including lysozyme concentration,temperature and lysis time,was studied by orthogonal design analysis,and the results indicated that the highest formation ratio of 99%was achieved when the lysozyme solution was 2mg/ml, incubated at 28℃for 90min.The protoplasts were plated on dry plates of R₂YE by spreading with a glass spreader,and the regeneration ratio was over 48%when cultured at 25.5℃.3.The gene transformation system of mutant UV2136 was developed by protoplasts transformation in the study.UV2136,the mutant blocked in cytosinpeptidemycin biosynthesis,was highly sensitive to thiostrepton,plasmid pIJ702 and pWHM3 containing thiostrepton resistance gene,tsr,were used to transform UV2136.Without endogenous plasmid,UV2136 owns strong restriction and modification systems which prevent the transformation of foreign plasmid.Many possible solutions were adopted,including heat attenuation,heat treatment,chill treatment,EDTA treatment and plasmid denaturation,no transformant was found when pIJ702 was used.Non-methylated pWHM3 derived from E.coli ET12567 also can not be transformed into UV2136,while the pWHM3 from methylatng host,E.coli DH5α,can be introduced at a low frequency,4 transformants perμg pWHM3,and the pWHM3 modified by UV2136 itself can be transformed at a higher frequency,10⁴ transformants perμg pWHM3.4.Conjugation was used to develop the transformation system of wild strain of cytosinpeptidemycin producing 04-2-2.To construct pKC1139-tsr,pIJ702 was digested by Belâ… ,the fragment of 1,055bp containing tsr gene was recovered and inserted into the pKC1139/Bglâ…¡which was dephosphorylated by CIAP.To construct pSET152-tsr,tsr gene was cloned by PCR,digested by ApaI and NheI and ligated with pSET152/ApaI/NheI.By conjugation,pKC1139-tsr can be transformed into the wild strain 04-2-2,while pSET152-tsr cannot be introduced,probably because there is no attP site which is necessary for the replication of pSET152-tsr. 5.By complementation of UV2136,three genes involved in the biosynthesis of cytosinpeptidemycin were cloned and function analyzed.Total DNA of the wild strain 04-2-2 was partially digested by EcoRI and Hindâ…¢individually and 3-15kb fragments were recovered,ligated with pWHM3/EcoRI and pWHM3/Hindâ…¢which was dephosphorylated by CIAP.The ligated product was introduced into UV2136 by protoplast transformation system and the cytosinpeptidemycin activity of thiostrepton resistant strains was determained. A transformant,UV2136-R,can produce cytosinpeptidemycin,and the insert was analyzed. There are three ORFs designated cytoA,cytoB and cytoC.cytoA,consisted of 1464bp, encodes a protein with 487 amion acid resdues.Database searching showed that CytoA belongs to HATPase_c superfamily,is homologous to sensor kianse from Streptomyces pristinaespiralis ATCC 25486 with 66%identities,suggesting that the expressed product of cytoA maybe histidine kinase.cytoB,consisted of 570bp,encodes a protein with 189 amino acid residues.Database Blasting showed that CytoB belongs to REC superfamily,is highy homologous to response regulator from Streptomyces griseus subsp.griseus NBRC 13350 with 86%identities,illuminating that CytoB is response regulator,consisting of two-competent system together with CytoA,and gene accession No.was GQ149124. Disruptant of cytoA/B could produce cytosinpeptidemycin,which indicated that cytoA/B was not related to the production of cytosinpeptidemycin directly,but was regulatory genes.cytoC, consisted of 783bp,encodes a protein with 260 amino acid residues.Database searing showed that CytoC belongs to DUF899 superfamily,is high homologous to conserved hypothetical protein which is thioredoxin-like from Frankia sp.ccI3 and Streptomyces pristinaespiralis ATCC 25486 with 85%identities.Disrupant of cytoC losing the ability to produce cytosinpeptidemycin,suggested that cytoC is an important gene involved in the biosynthesis of cytosinpeptidemycin.6.By homologous cloning,a biosynthetic gene named cytoD encoding glycosyltransferase was cloned and function analyzed.There are cytosinine in both cytosinpeptidemycin and Blasticidin S,it can be induced that there are similar genes involved in the biosynthesis.Using partial sequence of BlsD as probe,total digested genome DNA of 04-2-2 was Southern blotted,and a 2.1kb signal fragment was found.By construction of gene sub-library and colony in situ hybridization,a colony containing signal fragment was achieved and the plasimid was named pUC18-CGA.There is 1 ORF named cytoD,which is 981bp and the expressed product is a protein with 321 amino acid residues.Database blasting showed that CytoD is high homologous to glycosyltransferase from Streptomyces griseus subsp.griseus NBRC 13350 with 85%identities,suggesting that CytoD maybe glycosyltransferase.Disruptant of cytoD lose the ability to produce cytosinpeptidemycin, which illustrated that cytoD is an important gene involved in the biosynthesis of cytosinpeptidemycin.All the results were essential for the cloning of cytosinpeptidemycin biosynthetic genes and illustrating the total biosynthesis.